Introduction

Enzootic bovine leukosis is an economi- cally important disease of cattle which usual- ly causes clinically silent infections. The dis- ease occurs worldwide, infecting cattle and, regionally, water buffalo. Bovine leukemia virus (BLV) as the etiologic agent of the dis- ease is a single-stranded positive sense RNA delta-retrovirus, from the Retroviridae fam- ily. While about 70% of BLV infected  cat- tle remain asymptomatic (Aleukemic form), approximately 30% develop persistent lym- phocytosis (PL) with polyclonal  expansion of B lymphocytes. Only 1-3% of infections progress to the form of multicentric lym- phosarcoma (LS) (Maclachlan and Dubovi, 2010; Murakami et al., 2018; Okagawa et al., 2018; Ruggiero et al., 2018). Although few of the BLV infected cattle actually develop clinical signs of the disease, the infection poses economic losses relating to the de- creased longevity, reduced milk production and compromised immune system. Rat, rab- bit, pig, chicken, goat and sheep can be ex- perimentally infected by the virus, but cattle and water buffalo as the natural hosts of BLV are particularly important to epidemiologi- cal investigations (Maclachlan and Dubovi, 2010; Kuczewski et al., 2018).

Serological assays including en- zyme-linked immunosorbent assay (ELISA) as the most common detection methods for identifying BLV infected animals are rapid and inexpensive. However, as antibody re- sponse may not be induced until 14 weeks after infection, they are not always efficient. It has been demonstrated that BLV infected cattle could be transiently seronegative in some conditions such as early infections, per parturient state and co-infection with other viruses (Roberts et al., 1988). In such situa- tions while the animal is viremic and able  to

transmit the virus, the serology result is neg- ative (Kuckleburg et al., 2003). On the other hand, cross reactivity with other members of Retroviridae family could cause false posi- tive results. Molecular methods such as PCR and more sensitive; nested-PCR have pro- vided reliable diagnosis in early stages even in young animals and attracted much atten- tion in recent years (Gonzalez et al., 2001; Benavides et al., 2017).

The world population of water buffalo (Bubalus bubalis) is approximately 168 mil- lion head; more than 95 percent are located  in Asia (FAO, 2017). Buffalo is among the most productive domestic animals and has been recognized for thousands of years  as  an efficient draught species. As a result of unique conversion capacity, buffalo is con- sidered as a source of high quality meat which can be produced more cheaply than cattle. Buffalo can thrive under different cli- matic conditions and adapt to all save  arid or arctic environments. Under proper man- agement, the animal adjusts readily to inten- sive agricultural systems in high temperate climates (Cockrill, 1981).

The population of water buffalo in  Iran was recorded approximately  127000  head  in 2017 (FAO, 2017). Iranian buffaloes are commonly maintained in the south, north, northwest and southwest areas mainly for meat and milk purposes, playing a key role in the economy of rural families (Naserian and Saremi, 2007; Oryan et al., 2010). Although BLV is relatively prevalent in Iranian cattle, there is no information on its prevalence in water buffalo populations as another natural host of the virus. The aim of the study de- scribed here was to evaluate the occurrence and seroprevalence of BLV infection among water buffalo population in Khuzestan  prov-

ince, Southwest Iran.

Materials and methods

Sampling and DNA extraction

The study was performed on a total of 100 Iranian water buffalo (≤ 3 years old) referred to the slaughterhouse in Khuzestan province, southwest Iran from June 2016 to December 2017. Blood samples were collected  from  the cephalic veins and sera were separated and stored at –20 °C until serological assay. Blood samples with ethylene diamine tetra acetic acid (EDTA) were also collected for molecular analysis. DNA extraction from peripheral blood leukocytes was performed using a Genomic DNA Extraction Kit (Genet Bio, Seoul, Korea). The study was approved by Shiraz University, Policy on Animal Care and Use.

Enzyme-linked immunosorbent assay (ELISA)

For detection of antibodies against Bovine leukemia Virus in buffaloes’ sera  samples, the Enzyme-linked immunosorbent assay (ELISA) test was performed. Anti-gp51 BLV antibodies were detected using a commercial competitive ELISA kit (IDVet, France) ac- cording to the manufacturer’s instructions. The sample/negative control (S/N) percent- age was calculated by the formula:

S/N% = [ODsample] / [ODNC] ×  100.

Sera with an S/N% ≤ 50 were considered negative; samples with S/N% between 50 and 60 were considered doubtful; and sam- ples with S/N% ≥ 60% were considered positive.

Detection of BLV  provirus

Proviral BLV DNA was detected by nest- ed polymerase chain reaction of the BLV env gene. Two pairs of primers were used for PCR amplification as follows: External  primers  5'-     TCTGTGCCAAGTCTCCCAGATA-3'

and   5'- AACAACAACCTCTGGGAAGG-

GT-3' as well as internal primers 5'- CCCA- CAAGGGCGGCGCCGGTTT-3', and 5'- GCGAGGCCGGGTCCAGAGCTGG-3'

which resulted in the amplification of 598 and 444 bp DNA fragments in the gp 51 re- gion of the env gene, respectively.

Amplification was performed in a final volume of 25 µL containing 20 ng genom-  ic DNA, 100 ng of each primer, 1.5 mM MgCl2, 200 μM of each dNTP, 50 mM KCl and 20 mM Tris–HCl, and 1 U/μL Taq DNA polymerase (CinaClon, Iran). The thermal cycling profile was 1 cycle of 94 °C for 9 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 62 °C (for external prim- ers) or 70 °C (for internal primers) for 30 s and extension at 72 °C for 60 s, with a termi- nal extension of 4 min at 72 °C. The second round of PCR was carried out using 1 μl of the first-round product and the same concen- tration of reagents and corresponding prim- ers. The amplified PCR products were de- tected using electrophoresis on 1% agarose gel and visualized by ultraviolet (UV) light. BLV positive control was  kindly  provided by Tehran University, Iran. DNA sample prepared from confirmed BLV-seronegative cattle was also used as negative  control.

Statistical analysis

The data was presented in a 2×2 table and analyzed for sensitivity, specificity, and pre- dictive values using the following formulas: Sensitivity= number of true positives/ (number  of true  positives+  number  of false

negatives)

Specificity= number of true negatives/ (number of true negatives+ number of false positives)

Positive predictive value= number of true positives/ (number of true positives+ number of false positives)

Table 1. Sensitivity and specificity, positive and negative predictive values the ELISA against PCR

Negative predictive value= number of true negatives/ (number of true negatives+ num- ber of false negatives).

Results

Out of 100 samples collected from buf- faloes, 52 samples (52%) had antibodies against BLV gp51 protein, measured by ELI- SA technique. Nested PCR of gp 51 region in the env gene also revealed 47 positive spec- imens (47%), giving 598 and 444 bp DNA fragments.

ELISA yielded 52 positive suspects, 5 of them were PCR negative. According to the Table 1, the sensitivity, specificity, positive and negative predictive values of the ELISA were 100%, 90.56%, 90.38% and 100%,  re-

spectively.

Discussion

Of the 194.29 million buffaloes in the world, 179.75 million (92.52%) are water buffaloes, primarily located in Asia (Hamid et al., 2016). Water buffaloes are distributed in different parts of Iran, posing an indis- pensable source of human needs including meat and milk. Unfortunately, few studies have been conducted to investigate the vi-  ral pathogens infecting water buffaloes, es- pecially viruses that cause clinically silent infections while acting as a source of virus transmission. Results of the present study showed the BLV seroprevalence of 52% by ELISA and 47% by nested PCR in the water

buffalo population in the southwest of Iran. To the best of our knowledge, this is the first research that attempted to study the BLV in- fection in buffalo population in  Iran.

Limited studies have investigated the se- roprevalence of BLV in buffalo population  all around the world. Moreover, different serological and molecular methods with dif- ferent sensitivity and specificity levels have been applied to determine the disease status in buffalo populations, including ELISA, agar gel immunodiffusion (AGID), immu- nofluorescence (IF), viral neutralization test and PCR. A study conducted on 370 water buffaloes in Pakistan reported 0.8 % preva- lence of anti-BLV antibodies by immunodif- fusion test (Meas et al., 2000b). Mingala et al. also reported 27.6% prevalence in Phil- ippines water buffaloes obtained by nested PCR (Mingala et al., 2009). Oliveira et al. investigated the seroprevalence of BLV in 315 buffaloes from different regions in Bra- zil by commercial ELISA, AGID and PCR methods. In spite of detection of antibodies against the whole virus in 24.44% of serum samples by ELISA, the immunodiffusion test and PCR technique targeting the env and tax genes revealed negative results (De Oliveira et al., 2016). Investigation of BLV in cattle has also demonstrated that ELISA can give false positive results, when compared with direct detection test such as PCR. The prev- alence  of BLV  in cattle  in Thailand  ranged